Tryptophan is one of the essential amino acids constituting the bodies of animals, and is important as a medicine, nutrient, or as an additive for animal feed. Known methods for producing tryptophan include fermentation processes and enzymatic processes. For fermentation processes, tryptophan could be produced from its precursors either by adding anthranilate or indole with sugar to a culture, or by direct fermentation of glucose in the culture. For enzymatic processes, tryptophan could be produced from indole, pyruvic acid and ammonium ion by using a microorganism-produced tryptophanase. An enzymatic process has the advantage that a simpler process for purification and higher purity of tryptophan are achievable and such process has become a method of choice for producing the medicinal grade of L-tryptophan.
In the production of L-tryptophan using bacteria producing high levels of tryptophanase, one of the substrate indole is toxic to bacteria and causes substrate inhibition even at low concentration (18 mM). It is therefore disadvantageous to use the indole concentration as the driving force for the synthetic reaction. Also, tryptophan over 50 mM causes product inhibition and sodium pyruvate decomposes in the presence of NH4.sup.+ ion at alkaline pH (pH 9.0). As a result, methods of producing L-tryptophan at high concentration could be obtained by continuous or stepwise feeding of concentrated indole to maintain limiting concentration of indole, and with the help of tryptophan precipitant--inosine, higher concentration of L-tryptophan (&gt;80 g/L) is achievable.
There are many known methods for producing L-tryptophan using tryptophanase. Japanese Patent 56085291 describes production of L-tryptophan by reacting indole with serine or with pyruvic acid and ammonium ion using strain of genus Aeromonas, Vibrio or Bacillus. U.S. Pat. No. 4,349,627 describes a method wherein L-tryptophan is produced from indole and serine or from indole, pyruvic acid, and ammonium ion with a particular microorganism of genus Enterobacter. Tryptophanase producing strains including Proteus rettgeri, Proteus vulgaris, Proteus mirabilis, Proteus morganil and Escherichia coli were used to prepare L-tryptophan in Japanese Patent 62134094. Japanese Patent 63137689 describes using heat treated microorganisms including various species of Escherichia coli to prepare L-tryptophan from indole, pyruvic acid and ammonium ion.
Japanese Patent 1104186 describes a method of producing L-tryptophan from indole and L- or D,L-serine using tryptophan synthase. French Patent 2581654 describes a method of reacting indole with pyruvate and ammonium ion by immobilized tryptophanase and recovering L-tryptophan by precipitation with inosine. Japanese Patent 64051093 describes a method of preparing L-tryptophan using tryptophanase with stepwise feeding of indole to maintain its concentration below 4 mM and 39 mM of L-tryptophan can be produced in 10 hours of reaction. Japanese Patent 01191692 describes a process using immobilized cell to prepare L-tryptophan from indole and pyruvic acid wherein indole is continuously fed to maintain at 17 mM and L-tryptophan is continuously or stepwisely removed by a separator, 36.49 g of L-tryptophan can be obtained in 120 hours of operation from 1 L of reactor in which the fractional conversion of indole and pyruvate is 92% and 84%, respectively., European Patent 381744 describes a multi-stage process for the production of L-tryptophan where a bacterial host cell was transformed to include tryptophanase which was then used in a subsequent bioconversion stage to accumulate L-tryptophan by continuously controlled feeding of indole to maintain its concentration below 10 mM in a batch reaction and 24 g/L of L-tryptophan was produced in 75 minutes, which gave over 99% fractional conversion of indole.
Although inexpensive raw materials such as ammonia can be used to synthesize L-tryptophan with indole and pyruvate that are of economic advantages, there are always many serious problems in the process such as indole inhibition, L-tryptophan inhibition and decomposition of pyruvate. To date, there are some enzymatic processes that produce L-tryptophan in rather high yield and high rate by controlled feeding of indole and/or pyruvic acid, but there is no report concerning feeding of indole, pyruvate and NH.sub.4 Cl by on-line feedback control operated simultaneously.
In addition to the known problems, a phenomenon has been observed in this invention that except under optional conditions, tryptophanase will decay when it is incubated with two of the substrates--sodium pyruvate and NH.sub.4 Cl. In order to overcome the above problems, feeding of indole, sodium pyruvate, and NH.sub.4 Cl is controlled by a computer equipped with an on-line indole assay device of this invention. The computer controls the feeding of indole and NH.sub.4 Cl by a pre-defined profile that agrees with a tryptophanase activity profile. Sodium pyruvate is added at a predetermined time during the reaction. The product inhibition is overcome by addition of L-tryptophan precipitant--inosine which can remove L-tryptophan from reaction system through forming an insoluble complex with L-tryptophan. The combination of feedback control of substrate feeding together with the introduction of inosine into the reaction vessel results in a high efficient process for enzymatic synthesis of L-tryptophan.